Soil Biology & Biochemistry 75 (2014) 64e72

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Soil Biology & Biochemistry

journal homepage: www.elsevier.com/locate/soilbio

Assessing the impacts of chemical cocktails on the soil ecosystem

J. Horswell a, *, J.A. Prosser a, A. Siggins a, A. van Schaik a, L. Ying b, C. Ross c, A. McGill d,
G. Northcott e
a

Institute of Environmental Science and Research (ESR) Ltd, Kenepuru Science Centre, Porirua, New Zealand
Plant and Food Research Ruakura, Private Bag 3230, Hamilton, New Zealand
c
Landcare Research, Palmerston North, New Zealand
d
Landcare Research, Hamilton, New Zealand
e
Northcott Research Consultants Ltd, 20 River Oaks Place, Private Bag 3200, Hamilton, New Zealand
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 26 November 2013
Received in revised form
12 March 2014
Accepted 18 March 2014
Available online 13 April 2014

Little is known about the environmental fate and effect of low levels of co-contaminants that are
commonly present in wastes such as biosolids. Lysimeters were established using soils contaminated
with Cu or Zn and augmented with triclosan. Triclosan degraded rapidly in the soils, with methyl-triclosan being the major degradation product. However, as metal concentration increased, transformation
and biodegradation of triclosan decreased. For some soil health indicators (e.g. sulphatase enzyme),
results suggested that general toxicity was increased when metals and triclosan were both present. These
preliminary results suggest that co-contaminants can result in a combined effect that is potentially
greater than the sum of the individual effects, with additional impacts on the rate and extent of
contaminant degradation.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Biosolids
Triclosan
Heavy metals
Co-contaminants
Soil health indicators

1. Introduction
Triclosan (5-choloro-2-(2,4-dichlorophenoxy) phenol; TCS) is
a broad spectrum antimicrobial agent, which is used in a wide
variety of personal care products including deodorants, hand
soaps, toothpaste, textiles, laundry detergents, antiseptics,
shower gels and cleaning agents. Household products containing
triclosan are typically discarded into the sewage system. The level
of transformation and biodegradation of TCS in waste water
treatment plants (WWTPs) varies with operating conditions (Xia
et al., 2005). Some studies have shown >90% removal of TCS using activated sludge as secondary treatment (McAvoy et al., 2002;
Bester, 2003; Kanda et al., 2003; Sabaliunas et al., 2003; Heidler
and Halden, 2007). However, due to the hydrophobic nature (log
Kow 4.8) of TCS, it is likely that a signicant removal mechanism
is sorption onto biosolids (McAvoy et al., 2002; Reiss et al., 2002).
Both TCS and its transformation product methyl-TCS (5-chloro-2(2,4-dichlorophenoxy)-anisole) have been detected in surface
waters downstream of sewage treatment plants (Lindstrom et al.,
2002; Kookana et al., 2011). The limited data available in the

* Corresponding author. Tel.: 64 4 9140684; fax: 64 4 9140770.

E-mail address: Jacqui.Horswell@esr.cri.nz (J. Horswell).
http://dx.doi.org/10.1016/j.soilbio.2014.03.013
0038-0717/ 2014 Elsevier Ltd. All rights reserved.

literature suggests that the concentration of TCS in efuents can

range from 35 ng L1 to 2700 ng L1 (McAvoy et al., 2002; Reiss
et al., 2002; Singer et al., 2002; Sabaliunas et al., 2003; Halden
and Paul, 2005). In a study of 19 efuents in Australia, Kookana
et al. (2011), found concentrations of TCS ranging from 23 to
434 ng L1. A similar study of TCS in the inuent and efuent of 13
WWTPs in New Zealand found TCS concentrations ranging from
25 to 100 ng L1 in the inuent and 4.43e158 ng L1 in treated
efuents (Strong et al., 2010). Concentrations of TCS in biosolids
have been found to be an order of magnitude higher than in efuents ranging from 0.43 to 133 mg kg1 in the USA, (McAvoy
et al., 2002; USEPA, 2009; Cha and Cupples, 2009); from 0.09 to
16.79 mg kg1 in Australia (Kookana et al., 2011) and 1.05e
17.23 mg kg1 in New Zealand biosolids (Speir and Northcott,
2006).
A major pathway for the movement of organic contaminants
such as TCS to the environment is through the land application of
biosolids, a common practice in many countries (Lozano et al.,
2012). Triclosan and other broad spectrum antimicrobial chemicals are specically added to personal care products to prevent
their deterioration by microorganisms. TCS targets numerous
intracellular and cytoplasmic sites within cells, it may inuence the
transcription of genes participating in amino acid, carbohydrate
and lipid metabolism (Reiss et al., 2009).

J. Horswell et al. / Soil Biology & Biochemistry 75 (2014) 64e72

It is these features that can unintentionally affect soil and

aquatic animals should their habitats become contaminated with
such chemicals. In a soil environment, previous studies have
demonstrated inhibition of soil processes however, these are often
short lived and the microbial community recovers (Waller and
Kookana, 2009; Butler et al., 2011). Butler et al. (2011) measured
both basal and substrate-induced respiration (SIR) in three soils
types and found that TCS inhibited both parameters at concentrations as low as 10 mg/kg (in the loamy sand soil), however, both
basal respiration and SIR recovered. Waller and Kookana (2009)
also demonstrated TCS inhibited SIR in Australian sandy and clay
soils. Their study demonstrated that TCS at concentrations below
10 mg/kg disturbed the nitrogen cycle in some soils (sandy soil;
Waller and Kookana, 2009). Harrow et al. (2011) demonstrated
changes in microbial numbers and community structure after
irrigation of soils with greywater containing TCS. Impacts on microbial community structure were also found by Drury et al. (2013)
in articial streams.
Biosolids contain a suite of contaminants including heavy
metals, which can be present at concentrations signicantly higher
than organic compounds, primarily because they are not degraded
by WWTP processes. Among the most prevalent of heavy metals in
sewage are copper (Cu) and zinc (Zn), originating from both industrial and domestic sources (Smith, 1996). Copper and Zn
therefore are the elements most likely to limit the amounts of
treated sludge that can be applied to land (Smith, 1996). Heavy
metal contaminants, present a risk to biological components of soil
systems because of their persistence, toxicity to soil organisms and
impairment of biological functions (Brookes and McGrath, 1984;
Giller et al., 1998; Speir et al., 2007). What remains unknown is
whether low concentrations of numerous compounds in biosolids
combine to produce synergistic/antagonistic/additive ecotoxicological effects on ecosystems (Daughton and Ternes, 1999;
Daughton, 2003; Dorne et al., 2007).
As in other countries, current risk assessment procedures in
New Zealand are reductionist, focussing on the fate and effects of
individual chemicals and/or dened classes of chemicals in isolation from other contaminants present in biosolids. As a result, there
is incomplete information about the environmental fate and effects
of the complex cocktail of components in biosolids (e.g. nutrients,
biological and chemical contaminants). Taking a rst step to a holistic understanding of the toxicological effects and impacts of
complex mixtures of contaminants is challenging, but is critical to
assessing the potential risks that chronic low-level exposure may
present to the environment.
In this study we investigated the fate and effects of TCS in
combination with copper or zinc in soil. We selected TCS as a model
organic antimicrobial chemical to investigate the potential effects it
may elicit on soil microbial activity and function in combination
with the heavy metals Cu and Zn which are biologically active and
found at high concentrations in wastes such as biosolids.
An added complication to understanding the interaction of
complex mixtures of contaminants in biosolids was illustrated by
Speir et al. (2003, 2007) who postulated that responses of sensitive
soil health indicators (such as enzymes) to potentially toxic elements can be masked by the effects of added organic matter (such
as biosolids). Thus, in this study we avoided these confounding
effects by using eld soils historically contaminated with copper or
zinc salts, and amending these with TCS spiked at 5 mg kg1 and
50 mg kg1, in the absence of biosolids. The degradation dynamics
of TCS in the presence of increasing concentrations of heavy metals
was measured, as well as changes in the soil microbial community.
Effects of the co-contaminants on soil enzymes (phosphate and
sulphatase), biomass and respiration, Most Probable Number
Rhizobium, and the activity of sensitive microbial biosensors (Lux)

65

were measured as well as terminal restriction fragment length

polymorphism (T-RFLP) bacterial community analysis.
2. Materials and methods
2.1. Experimental background and sample site
The soil was obtained from an existing eld trial, on a Horotiu
sandy loam (a Vitric Orthic Allophanic Soil, Vitric Hapludand) at
Ruakura, Hamilton, New Zealand (175 190 E, 37 470 S) (established
April 2007). Soil physiochecmial properties are show in Table 1.
The trial comprised 30 plots (1 m2), set out in two fullyrandomised replicate blocks. Each block contained seven plots
amended with Cu, seven plots amended with Zn and a common
unamended control plot. Prior to amendment, the top 100 mm of
soil over the entire 1 m2 plot was removed and sieved (6.5 mm) to
break up large clumps and remove vegetation (pasture species),
then thoroughly mixed in a concrete mixer. Sulphate salts of Cu
(CuSO4) or Zn (ZnSO4) were added to the mixing soil to raise metal
concentrations and a basal dressing of superphosphate, (NH4)2SO4
and KCl was also mixed in at the same time. The amended soil was
returned to its plot and packed down to its original eld density.
The plots were sown with a ryegrass/inoculated clover pasture seed
mix. In 2010, three years after the trial was set up, approximately
15 kg of soil was collected from 11 of the plots to a depth of 10 cm
sieved (2 mm) and stored at 4  C until required. The concentrations
of Zn and Cu in the soils used for the current experiment are presented in Table 2.
2.2. Lysimeter construction
Lysimeters were established in duplicate, with 33 treatments
resulting in a total of 66 lysimeters. Each treatment contained a
combination of varying concentrations of TCS and one of the heavy
metals. Specically, a range of ve metal concentrations (Cu 101,
187, 478, 1083 and 2944 mg kg dry soil1; Zn 186, 283, 632, 1224
and 2235 mg kg dry soil1; Table 1). The range of metals were
chosen in order to obtain high enough Cu and Zn concentrations in
soil to sufciently inhibit soil health indicators measured in this
study (e.g. biomass and respiration) and allow accurate determination of dose response curves (covering previously determined EC
50 values for Cu and Zn, Speir et al., 2007). The metal spiked soils
were combined with either 0, 5 (low) or 50 (high) mg kg1 TCS.
Three control treatments containing no (or only background levels
of) heavy metals, combined with 0, 5 or 50 mg kg1 TCS were also
included in the study.
The lysimeters were constructed from 15 cm lengths of PVC pipe
( 13 cm). The base of the lysimeters contained a layer of gravel to
retain the soil and assist drainage. Field moist sieved soil (1 kg) was
adjusted to 65% water-holding-capacity (WHC) and spiked with
TCS to obtain a nal concentration of 0, 5 or 50 mg kg1 TCS. The
spiking procedure was adapted from Brinch et al. (2002) to minimise the impact of organic solvents upon soil microbes. Briey,
250 g eld moist soil was distributed evenly on the base of a large
shallow glass container. Either 3.56 mg or 35.57 mg of triclosan (for
the 5 and 50 mg kg1 spiked soils respectively) was weighed into a
glass vial and mixed with 20 mL of acetone. Using disposable glass
Pasteur pipettes, the TCS-acetone solution was dripped evenly over

Table 1
Selected physiochemical properties of the Horotiu sandy loam.
Total carbon (%)

Total Nitrogen (%)

pH (H2O)

CEC (cmol kg1)

11.6

5.4

39

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J. Horswell et al. / Soil Biology & Biochemistry 75 (2014) 64e72

Table 2
Total Cu and Zn conc. of soils as analysed immediately prior to lysimeter
construction.
Treatment

Cu (mg kg dry soil1)

Zn (kg dry soil1)

Zero
Cu1
Cu2
Cu3
Cu4
Cu5
Zn1
Zn2
Zn3
Zn4
Zn5

30
101
187
478
1083
2944
29
29
31
28
30

136
109
109
106
96
87
186
283
632
1224
2235

the surface of the 250 g portion of soil. After through mixing, the
soil was left in the dark at room temperature overnight (16 h) to
evaporate residual acetone. The TCS spiked soil was then mixed
with the remaining 750 g of soil and packed into the lysimeters. The
lysimeters were placed in a dedicated outdoor lysimeter facility and
seated within a bed of pea gravel to within 1e2 cm of the top. The
lysimeters were sown with ryegrass and irrigated when insufcient
rainfall was observed. After 6 months, the lysimeters were harvested and analysed for a variety of chemical and biological indices.
2.3. Chemical and biological analyses
Total Cu and Zn were measured on pressed discs of nely ground
air-dry soil using X-Ray Fluorescence (XRF) spectrometry. Soil solution was extracted using the centrifugation method of Gillman
(1976). After centrifugation, the resulting ltrate (soil solution)
was collected and analysed for heavy metals by Inductively Coupled
Plasma Mass Spectrometry (ICPMS). Soil pH and total C were
determined according to Blakemore et al. (1987). Moisture content
was determined by overnight drying at 105  C.
Triclosan and degradation/transformation residues were
extracted from the soil using Accelerated Solvent Extraction (ASE)
with a mixture of dichloromethane/acetone (1:1) at 1500 psi and
100  C for 5 min. Each cell was extracted with two static cycles
followed by a ush volume of 60% with the same solvent mix. The
solvent extract was evaporated to dryness under a stream of nitrogen gas (Zymark Turbo-Vap) and redissolved in dichloromethane/methanol (95/5). An aliquot of the extract was cleaned up
using orosil adsorption chromatography (Biotage Isolute 1 gm),
evaporated to dryness, and derivatised by silylation using MTBSTFA
and heating to 60  C for 30 min. Calibration standards containing
the target compounds and internal standards were derivatised with
each batch of individual samples. The resulting trimethylsylil esters
were analysed by high resolution gas chromatography massspectrometry using an Agilent 6890N gas chromatograph (GC)
coupled to an Agilent 5975A inert XL mass spectrometer (MS) and
CTC autosampler. A 1 ml volume of derivatised sample extract was
injected into an Agilent split/splitless injector at 280  C with a
splitless time of 45 s and the components separated on a Varian
Factor Four capillary column with 5% phenyl phase and integrated
retention gap (40 m length; 0.25 mm lm thickness; 0.25 mm ID)
using a constant ow rate of helium (1 ml min-1). The column was
held at 90  C for 1.5 min, increased at 20  C min1 to 150  C, with a
second increase of 8  C min1 to 234  C, followed by a 6  C min1
increase to 280  C, and nally increasing at 20  C min1 to 330  C
with a 5.5 min hold. The mass spectrometer interface temperature
was maintained at 280  C, and ion source and quadrupole at 230  C
and 150  C respectively. Compound specic mass fragments were
obtained using single ion monitoring and were used in

combination with retention time matching for positive identication. The concentration of the three target analytes (TCS, methylTCS, and 2,4-dichlorophenol) was determined by extracting compound specic mass ions and comparing the relative abundance of
the four mass ions against those obtained from pure compound
standards. Quantication of target analytes was completed by internal standard quantitation using Agilent MSD Enhanced Chemstation quantitation software.
Soil basal microbial respiration was determined by the gaschromatographic method of Sparling et al. (1986), using 12 g
fresh weight soil and incubating for 7 d. Microbial biomass C was
determined by the fumigation-extraction method of Vance et al.
(1987), after adjusting the soil to 60% WHC, and using the modications suggested by Sparling et al. (1990). Phosphatase and sulphatase enzyme activities were measured as reported by Speir et al.
(1984), except that all assays contained 0.5 g soil, and phosphatase
incubations were for 1 h only.
Most-probable-numbers (MPN) of Rhizobium leguminosarum bv.
trifolii were determined as in Speir et al. (2004) using the method
rst described by Vincent (1970). The bacterial bioassay, consisting
of a lux-modied Escherichia coli biosensor, was conducted using
the extracted soil solutions as described in Horswell et al. (2006).
2.4. Calculation of ecological dose parameters
Soil biological properties, rhizobial MPN and lux-biosensor responses were related to total Zn and Cu concentrations using the
sigmoidal dose response equation developed by CSIRO, Australia
(Barnes et al., 2003; Smolders et al., 2004), based on the model
developed by Haanstra et al., 1985. The equation is:

C
1 eHlog XlogEC50

where Y is the biological activity; C is the calculated maximum

value of Y; X is the metal concentration; EC50 is the metal concentration at which the biological activity is inhibited by 50%; and H
is the Hill slope. Using this model, values were obtained for both
EC50 and EC20 or Cu and Zn (mg kg dry soil1).
2.5. Terminal restriction fragment length polymorphism (T-RFLP)
Total genomic DNA was extracted from 0.3 g of each of the 66
soil samples using the Mo Bio Powersoil DNA Isolation Kit,
following the manufacturers guidelines. DNA was eluted in 50 ml of
Solution C6. The presence of genomic DNA was conrmed by
agarose gel electrophoresis (2%) and quantied using a Quant-iT
PicoGreen dsDNA kit (Invitrogen).
Bacterial 16S rRNA genes were amplied from each sample in
triplicate with forward primer 63F (50 -CAG GCC TAA CAC ATG CAA
GTC-30 ; Marchesi et al., 1998) and reverse primers 1087R (50 -CTC
GTT GCG GGA CTT AAC CC-30 ; Hauben et al., 1997), which were
labelled at the 50 end with the phosphoramidite dyes 6-FAM and
VIC, respectively. PCRs were performed in 50 ml reactions containing: 5 ml 10  NH4 reaction buffer ((NH4)2SO4, 16 mM; TriseHCl [pH
8.8 at 25  C], 67 mM; 0.01% Tween-20); 3 mM MgCl2; 200 mM of
dNTP (dATP, dTTP, dGTP, dCTP); 10 pmol of each primer; 0.5 U Taq
polymerase and 2 ml of a 1:20 (v/v) dilution of extracted DNA in
dH2O. The PCR reactions were carried out under the following
conditions: Initial denaturation at 94  C for 3 min, then 30 cycles of:
94  C 30 s, 55  C 30 s, 72  C 60 s, followed by a nal elongation step
of 72  C for 20 min. Negative controls containing no DNA were used
to screen for contamination. PCR products were visualised by UV
excitation after electrophoresis on a 2% agarose gel.

J. Horswell et al. / Soil Biology & Biochemistry 75 (2014) 64e72

Triplicate PCR products were pooled during purication to

reduce amplication bias and increase the DNA yield. Products
were puried using the QIAquick PCR purication kit (QIAGEN),
and quantied using a Quant-iT PicoGreen dsDNA kit (Invitrogen). Two hundred ng of each PCR product was digested with 20
U MspI in a nal volume of 30 ml, containing 0.1 mg mL1 acetylated
BSA (all Promega, UK). Restrictions were carried out in the manufacturers recommended buffer at 37  C for 3 h, followed by inactivation of the enzyme at 65  C for 20 min and storage at 4  C.
Purication of restriction fragments was carried out using MinElute Reaction Cleanup kit (QIAGEN).
The resulting uorescent 50 and 30 terminal restriction fragments (T-RFs) were quality checked by agarose gel electrophoresis
before being sent for terminal fragment analysis (Massey Genome
Service, Palmerston North, New Zealand) using an ABI3730 Genetic
Analyser (Applied Biosystems, UK). The internal size standard LIZ
GS500 (250) was added by the sequencing facility prior to peak
quantication. The T-RFLP prole data was collated and analysed
using the GeneMapper software v4.0. Fragments were included in
further analysis if they were between 50 and 500 base pairs in
length and were greater than 100 uorescent units. The relative
abundance of a T-RF in a prole was calculated as a proportion of
the total peak height of all the T-RFs in that prole, and T-RFs that
contributed less than 0.5% of the total peak height of a sample were
also excluded from further analysis. Relative abundances for
duplicate samples were averaged and these values were analysed
using the PRIMER 6 (Plymouth Marine Laboratory) statistical
package. After data were square-root transformed to satisfy assumptions of normality, they were subjected to non-metric
multidimensional scaling (NMS), using a BrayeCurtis similarity
matrix. Cluster analysis was performed on the T-RFLP data to
identify groups of samples with 40, 60 and 80% similarity.
2.6. Statistical analysis
The means and standard errors of ecological data were calculated using Excel 2012. Analysis of variance was performed using
SPSS and the signicance of difference assessed using Tukey posthoc tests at a 5% condence level (p  0.05).
3. Results

67

Fig. 1. Concentration of triclosan, methyl-triclosan and 2,4-dichlophenol (mg kg1)

remaining after 6 months eld ageing in the soils spiked with 5 mg kg1 triclosan and
zinc at varying concentrations. Error bars represent standard errors. * indicates signicant difference from control (p < 0.05).

3.2. Impacts of triclosan on soil health indicators

Some of the measured biochemical properties demonstrated a
signicant (p < 0.05) impact upon exposure to TCS when combined
with Cu or Zn (Figs. 5 and 6). This was particularly evident for
sulphatase enzyme activity, which exhibited a signicant reduction
in the presence of TCS when combined with even the lowest concentration of Cu in soil (101 mg kg dry soil1) (Fig. 5). By comparison phosphatase enzyme activity was not signicantly impacted
by the presence of Cu until a tipping point, or threshold concentration was reached, upon which enzyme activity rapidly declined
(Fig. 5). These results strongly suggest a possible synergistic effect
of the presence of co-contaminants, highlighted by the fact that
enzymes activity was not signicantly reduced in the presence of
TCS alone (Figs. 5 and 6) (control treatments). For the remaining
soil health parameters (basal microbial respiration, microbial
biomass C, rhizobial MPN and lux-biosensor) the presence of TCS
caused either insignicant (large statistical error), or no effects
above that which was evident from the presence of Cu and Zn
alone.

3.1. Biodegradation of triclosan in soils

Figs. 1e4 show the concentration of TCS, TCS methyl-ether and
2,4-dichlophenol remaining in lysimeter soils following 6 months
ageing under eld conditions. In the control soils containing either
5 or 50 mg kg1 TCS and no metals, TCS was rapidly degraded and
<10% of the parent compound remained in the soils after 6 months.
Rapid degradation of TCS was observed in all treatments, however,
a distinction was observed at high metal concentrations. The major
degradation product found in the soils was methyl-TCS (Figs. 1e4).
As the soil concentration of both Zn and Cu increased, there was a
reduction in both transformation and degradation of TCS in the
soils. This was most evident for the highest Zn and Cu treatments,
where signicantly higher concentrations of TCS remained in soil
compared to the control soils in three out of the four TCS treatments (Figs. 1, 3 and 4), and signicantly less methyl-TCS was
formed in the 50 ppm TCS treated soils (Figs. 2 and 4). Signicantly
less methyl-TCS was formed in the Zn 50 ppm TCS lysimeter soils
above a Zn concentration of 283 mg kg dry soil1 (Fig. 2) and the
concentration of 2,4-dichlophenol was signicantly reduced by the
presence of Zn in the Zn 5 ppm TCS treated soils compared to the
control soil (Fig. 1).

Fig. 2. Concentration of triclosan, methyl-triclosan and 2,4-dichlophenol (mg kg1)

remaining after 6 months eld ageing in the soils spiked with 50 mg kg1 triclosan and
zinc at varying concentrations. Error bars represent standard errors. * indicates signicant difference from control (p < 0.05).

68

J. Horswell et al. / Soil Biology & Biochemistry 75 (2014) 64e72

Fig. 3. Concentration of triclosan, methyl-triclosan and 2,4-dichlophenol (mg kg1)

remaining after 6 months led ageing in the soils spiked with 5 mg kg1 triclosan and
copper at varying concentrations. Error bars represent standard errors. * indicates
signicant difference from control (p < 0.05).

3.3. Ecological dose parameters

Effective concentrations that caused a 20% and 50% decline in
activity (EC20 and EC50) were calculated for each metal, at all TCS
concentration (0, 5 and 50 mg kg1; Tables 2 and 3). This analysis
was undertaken to determine if the presence of an organic cocontaminant, in this case TCS, caused a reduction in the relative
EC20 and EC50 for Zn or Cu, which would indicate increased toxicity
as a result of exposure to a mixture of co-contaminants.
Tables 3 and 4 show the Cu and Zn EC20 and EC50 values,
calculated from the CSIRO dose response model using total metal
concentrations of the soil samples. Most of the data demonstrated a
good t for the CSIRO model (high R2) and reliable EC values could
be determined (e.g. lux biosensor, phosphatase and sulphatase),
however some data did not exhibit a reasonable t to the model
(e.g. microbial biomass, respiration and rhizobium MPN). There are
several possible explanations for this and these have been discussed at length by Speir et al. (2008). The most likely explanation
with respect to this data set is the high variability within the data
combined with small number of eld replicates. As a result EC

Fig. 5. a) Sulphatase and b) Phosphatase enzyme activities in relation to total soil copper
treatment in soil spiked with 0, 5 and 50 mg kg1 TCS. Error bars represent standard
error. Values shareing the same letter are not signicantly different (p < 0.05).

values can be calculated, but condence intervals (statistical errors)

are large.
For the soils containing metals only, the calculated EC values
were greater than the NZ Biosolids Guidelines soil limit concentration of 100 mg kg1 for Cu and 300 mg kg1 for Zn (NZWWA,
2003). However, there are instances where EC20 and/or EC50
values are close to this limit (sulphatase activity, microbial biomass,
rhizobium MPN and the lux biosensor, Tables 3 and 4). In the soil
treatments containing metals in combination with TCS some of the
EC values decreased. For example the EC20 calculated for both sulphatase enzyme activity and the lux biosensor fell below the
guideline limit for Zn when in the 50 mg kg1 TCS soil treatment
(Table 4). For most of the measured soil health indicators, the
presence of the co-contaminant TCS did not signicantly impact the
EC50 value. One exception was the lux biosensor, where soil solution
extracted from Zn lysimeter soil spiked with 50 mg kg1 TCS, produced EC50 values signicantly lower than the control soils (Table 4).
In the Cu amended lysimeters, rhizobium MPN numbers in soil also
appear to be reduced in the presence of increasing TCS, however the
condence intervals (statistical errors) vary greatly (Table 3).
3.4. The effect on microbial community structure

Fig. 4. Concentration of triclosan, methyl-triclosan and 2,4-dichlophenol (mg kg1)

remaining after 6 months eld ageing in the soils spiked with 50 mg kg1 triclosan and
copper at varying concentrations. Error bars represent standard errors. * indicates
signicant difference from control (p < 0.05).

3.4.1. Terminal restriction fragment length polymorphism (T-RFLP)

A total of 172 different T-RFs were identied from the 33
samples after removal of peaks that did not meet the selection
criteria previously outlined. Of these, 97 were from the 6FAM
labelled 50 end of the gene fragment, with between 28 and 42 T-RFs
present in each sample. The remaining 75 T-RFs were from the VIC
labelled 30 end, with 27e42 detected in each sample.

J. Horswell et al. / Soil Biology & Biochemistry 75 (2014) 64e72

69

Table 4
The effective concentration (mg kg dry soil1) of Zn that caused 50% (EC50) and 20%
(EC20) decline in activity of six soil health indicators, with and without TCS (at either
5 or 50 mg kg1). Values indicated with different letters were determined to be
signicantly different (p < 0.05). EC values noted with * are indicated to be below or
close to the NZ guideline limits for soil Zn.
Property
Phosphatase

Fig. 6. Sulphatase enzyme activities in relation to total soil zinc treatment in soil
spiked with 0, 5 and 50 mg kg1 TCS. Error bars represent standard error. Values
shareing the same letter are not signicantly different (p < 0.05).

Non-metric multidimensional scaling (NMS) analysis of T-RFLP

relevant abundance proles revealed that the bacterial community
structure formed two distinct groups (A & B; Fig. 7). Statistical
comparison of these two groups by ANOVA (SPSS) showed that
they were signicantly different (p < 0.05). The soil samples containing the highest level of copper (2944 mg kg1) grouped
together (group A), while the remaining samples formed a distinct
separate group (group B). Samples within each group demonstrated >80% similarity, while between 40 and 60% similarity was
observed between the two groups (Fig. 7). The data did not indicate
any combined effect of TCS and metal.
4. Discussion
4.1. Biodegradation of triclosan in soils containing Cu and Zn
In all of the control treatments (no Cu or Zn) less than 10% of TCS
remained in soil after 6 months. This was not unexpected due to the
rapid degradation of TCS in soils- for example Kookana et al. (2011)
and Liu et al. (2009) reported half-life of only 18 days for TCS in
biologically active soils. In our study, the degradation of TCS was
signicantly reduced in the presence of Cu and Zn. At Cu concentrations of 2944 mg kg1, 29 and 26% of the applied TCS remained
in soil after six months eld ageing (5 mg kg1 and 50 mg kg1
Table 3
The effective concentration (mg kg dry soil1) of Cu that caused 50% (EC50) and 20%
(EC20) declines in activity of six soil health indicators with and without TCS (at either
5 or 50 mg kg1). EC values noted with * are indicated to be below or close to the NZ
guideline limits for soil Cu.
Property
Phosphatase

Treatment

0 ppm TCS
5 ppm TCS
50 ppm TCS
Sulphatase
0 ppm TCS
5 ppm TCS
50 ppm TCS
Lux biosensor
0 ppm TCS
5 ppm TCS
50 ppm TCS
Microbial Biomass 0 ppm TCS
5 ppm TCS
50 ppm TCS
Respiration
0 ppm TCS
5 ppm TCS
50 ppm TCS
Rhizobium MPN
0 ppm TCS
5 ppm TCS
50 ppm TCS

EC50

EC20

R2

Lower 95% Upper 95%

1373
1355
1123
2092
1180
2480
1912
2033
2014
2179
922
1394
3226
3071
No t
162
108*
46*

623
683
755
1133
308
2232
1483
1639
1181
1041
41*
510
2634
2695
e
145
82*
5*

0.965
0.971
0.962
0.979
0.969
0.869
0.997
0.997
0.998
0.858
0.766
0.891
0.685
0.674
e
0.856
0.928
0.741

991
1014
855
1756
630
1626
1683
1691
1913
1188
2
648
577
7
e
81
76
0

1903
1811
1474
2493
2208
3783
2173
2444
2120
4000
359,978
3000
18,029
1,360,688
e
325
152
87,716

Treatment

0 ppm TCS
5 ppm TCS
50 ppm TCS
Sulphatase
0 ppm TCS
5 ppm TCS
50 ppm TCS
Lux biosensor
0 ppm TCS
5 ppm TCS
50 ppm TCS
Microbial Biomass 0 ppm TCS
5 ppm TCS
50 ppm TCS
Respiration
0 ppm TCS
5 ppm TCS
50 ppm TCS
Rhizobium MPN
0 ppm TCS
5 ppm TCS
50 ppm TCS

EC50

EC20

R2

Lower 95% Upper 95%

1900
3793
2818
2718
2488
3258
446a
511a
331b*
No t
No t
449
No t
No t
No t
No t
No t
134*

716
1640
1401
961
711
257*
388a
350a*
227b*
e
e
78*
e
e
e
e
e
103*

0.930
0.836
0.900
0.884
0.877
0.831
1.000
0.998
1.000
e
e
0.821
e
e
e
e
e
0.826

1258
1419
1710
1399
1214
0
358
465
315
e
e
19
e
e
e
e
e
37

2870
10,133
4644
5277
5100
22,355,157
554
561
347
e
e
10,703
e
e
e
e
e
480

treated soils, respectively). This is likely due to inhibition of microbial degradation and transformation of TCS caused by the
presence of heavy metals in the soil. Previous studies have noted
that TCS degradation is signicantly reduced (virtually eliminated)
in sterile soil, highlighting the importance of microbial processes in
the breakdown of TCS (Ying et al., 2007; Kookana et al., 2011). We
hypothesise that the degradation and/or transformation of TCS has
been inhibited in these soils where the microbial community has
been previously compromised by prior exposure to the heavy
metals Cu and Zn.
The observed decrease of TCS in our lysimeter soil samples was
mirrored by an increase in the concentration of the bacterial
transformation product methyl-triclosan. The primary mechanism
of degradation of triclosan in this study was biotransformation as
microbially mediated degradation is the only known source of
methyl-triclosan (Lindstrom et al., 2002). This is consistent with the
results of Butler et al. (2012a) who identied methyl-triclosan to be
the predominant metabolite resulting from the biodegradation of
TCS in soils receiving biosolids. In our study the high Cu and Zn
lysimeter soil treatments spiked with 50 mg kg1 TCS contained
signicantly lower concentrations of methyl triclosan compared to
the control soil (Figs. 2 and 4). This result provides further evidence
that the presence of high concentrations of Cu and Zn in the
experimental soil inhibited the ability of the soil microbial community to metabolise TCS to the corresponding methyl ester.
Given that previous research has demonstrated signicant effects of TCS on biological parameters at levels as low as 10 mg kg1
(Butler et al., 2011) it is of signicant concern that we had levels of
TCS up to 13 mg kg1 remaining in Cu contaminated soils and up to
9 mg kg1 of TCS in Zn contaminated soils after six months. Indeed,
Amorim et al. (2010) found that the reproduction EC10s for three
soil invertebrate species were 0.6e7 mg TCS kg1 dry soil. Soil invertebrates were not investigated in our study, but the levels of TCS
remaining in our soil samples were frequently higher than these,
particularly those containing higher levels of Cu or Zn.

4.2. Effects of co-contaminants on soil health indicators

The soil health indicators chosen for assessment are wellaccepted properties used to measure the environmental impact of
soil contaminants (Speir et al., 2003). The addition of TCS to Zn and

70

J. Horswell et al. / Soil Biology & Biochemistry 75 (2014) 64e72

Fig. 7. NMS analysis plot of T-RFLP relative peak height data, calculated using the BrayeCurtis similarity index. A and B are two statistically signicant clusters (p < 0.05).

Cu contaminated soils signicantly reduced the activity of sulphatase and phosphatase enzymes. These reductions were most
obvious for sulphatase enzymes, which exhibited a signicant
reduction in activity in soils containing Cu and TCS at relatively low
levels (5 mg kg1 TCS, 187 mg kg1 Cu). However, TCS had no signicant impact on the remaining soil properties above that which
was observed from the presence of the metals alone. This may be
due to the rapid degradation of TCS as mentioned previously,
allowing many soil properties to recover over the 6 month experimental period. Previous studies have demonstrated inhibition of
soil processes at TCS concentrations similar to those used in this
study, however, these were often short lived and the microbial
community recovered (Liu et al., 2009; Waller and Kookana, 2009;
Butler et al., 2011). TCS has been previously demonstrated to inhibit
both basal and substrate-induced respiration (SIR) (Liu et al., 2009;
Waller and Kookana, 2009; Butler et al., 2011) in soil at concentrations as low as 10 mg kg1 and Liu et al. (2009) observed that TCS
signicantly inhibited soil respiration at TCS concentrations
exceeding 10 mg kg1 (dry soil) during the rst 4 days of incubation, but recovered after longer incubation. TCS disturbed the soil
nitrogen cycle in an Australian sandy soil at concentrations below
10 mg kg1 (Waller and Kookana, 2009).
The effect of TCS on soil enzyme activity is less denitive. Waller
and Kookana (2009) did not observe any adverse effect of TCS on
the activity of four soil enzymes (acid and alkali phosphotase, bglucosidase and chitinase). Liu et al. (2009) observed an initial
impact of TCS on phosphatase activity at concentrations between
0.1 and 50 mg kg1 dry soil but this recovered after 2 days after
which only minimal effects were observed.
Given that our experiment did nd an adverse effect of TCS on
sulphatase and phosphatase activity it may be related to the combination of metal and TCS together. The adverse effect we observed

on soil sulphatase and phosphatase activity in our experiments

occurred at concentrations of TCS that were within the range of
previous studies (Liu et al., 2009; Waller and Kookana, 2009; Butler
et al., 2011). The effects may be explained by combined impact of
metals and TCS upon the soil microbial community, and the
considerable difference in the incubation period between previous
experiments (one to two weeks, Liu et al., 2009; Waller and
Kookana, 2009) compared to the six month period of ageing in
our study.
Whilst some trends relating to the presence of TCS were
observed in other soil properties measured in this study, these were
not statistically signicant. These trends may be better understood
by carrying out further trials using a greater number of eld replicates and including higher concentrations of TCS.
4.3. Ecological dose parameters
For some of the soil health indicators we measured (sulphatase,
MPN Rhizobium and lux biosensor) there was evidence that the
presence of TCS as a co-contaminant acted to reduce the EC50/20
values for Zn and/or Cu to concentrations below those specied
within the NZ Biosolids Guidelines.
The nitrogen-xing bacterium R. leguminosarum bv. trifolii is an
economically vital component of New Zealands pastoral agriculture and indications that 50% of rhizobial viability (or even 20%)
may be lost at Cu and or Zn concentrations around the current soil
limit (NZWWA, 2003), and that this may be further reduced by the
presence of TCS as co-contaminant is of concern. It is important to
remember that the soils were amended with soluble metal salts, a
potential worst case scenario that would directly expose soil organisms to a relatively higher proportion of bioavailable metal.
However, the soils used in the current experiment were sampled

J. Horswell et al. / Soil Biology & Biochemistry 75 (2014) 64e72

three years after spiking, and it is likely that substantial proportions

of the metals would have become bound to the soil and less
bioavailable over time. There is evidence that this occurs when
biosolids are spiked with metals salts (McLaren and Clucas, 2001),
although the organic fraction of biosolids would be higher than in
soil the same process would be likely to occur.
4.4. The effect of triclosan-metal co-contaminants on microbial
community structure
Analyses of microbial community structure using T-RFLP did not
indicate any effect of TCS or TCS e heavy metal combined. The
biggest inuencing factor on microbial community structure
appeared to be the highest Cu treatment, which grouped independently from the remaining samples based on NMS analysis. This
result is in line with that of Butler et al. (2012b) who found that soil
type and time were the most explanatory factors effecting microbial community structure in their experiment. In their study they
found evidence of an effect of TCS on phenotypic responses, however, this was effectively masked by the inuence of soil type.
4.5. Implications for environmental guidelines
Although some research is available on the effects of TCS on soil
ecological functions, little is known about the potential effect of low
concentrations of numerous inorganic and organic chemicals present in biosolids (or other organic wastes) entering the soil environment. The results from this study have given valuable insight to
the potential effect of TCS when combined with Cu or Zn on soil
biological functioning.
It has been shown that the mineralisation of TSC in soil is
dependent on many factors, including soil chemistry, the existing microbial population, presence of oxygen, moisture, temperature and the form of the applied biosolids (Ying et al., 2007;
Al-Rajab et al., 2009; Kookana et al., 2011; Butler et al., 2012a).
Previous studies have found that the addition of biosolids to soil
reduces the transformation of TCS (Kwon et al., 2010). Given
that the current experiments were carried out without biosolids,
this may pose a further risk of the accumulation of TCS in soil
over time, particularly where co-contaminants are present and
exert an inhibitory effect upon biodegradation/transformation
processes. This raises concerns regarding the recycling of
contaminant containing biosolids to land. Current risk assessment procedures focus on the fate of individual chemicals and
do not consider the cumulative risk presented by mixtures of
multiple low level co-contaminants. Future guidelines should
acknowledge and take into account for the combination of
chemicals present in organic wastes and develop new risk
assessment procedures that incorporate thresholds for mixtures
of chemical contaminants.
5. Conclusions
This study has shown that the presence of heavy metal cocontaminants in soil can affect the microbial communities ability
to degrade TCS and therefore increase its persistence and potential
impacts on terrestrial organisms such as soil microbes. The preliminary data we have obtained from this experiment suggests the
presence of co-contaminants in complex waste materials such as
biosolids may combine to produce synergistic or additive ecotoxicological impact upon soil function and health indicators. The
longer term impact of the combination of heavy metals and TCS
upon these measures of soil function remains to be determined and
will be the subject of future experiments.

71

Acknowledgements
The New Zealand Ministry of Business, Innovation and
Employment (MBIE) (C03X0902) for funding this research
programme.
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